Our goal is to better understand how Epstein-Barr virus infects humans in order to develop more effective therapeutics that prevent or treat EBV-induced diseases. Specifically, this project focuses on how neutralizing antibodies that block either EBV infection of B or epithelial cells impact acute and persistent infection of the host aftr oral transmission. EBV infects B cells and epithelial cells through different viral proteins and receptors, but which cell types are critical for the initial phases of oral infection and viral amplification are unknown. More importantly, neutralizing B cell infection is being pursued as the basis for an EBV vaccine with little pre-clinical or clinical data to support the concept that neutralizing antibodies against EBV infection of B cells will have any impact on oral virus challenge. We use neutralizing antibodies as a probe in the most accurate animal model for EBV infection to ask whether this arm of the immune response alone is sufficient to disrupt acute and persistent infection upon oral virus challenge. 72A1 is the most studied of only a few known EBV neutralizing monoclonal antibodies (mab), and it binds the EBV major membrane glycoprotein, gp350, to inhibit EBV attachment to B cells. In order to use 72A1 in the rhesus macaque animal model for EBV infection, we replaced the native gp350 gene in the rhesus lymphocryptovirus (rhLCV) with EBV gp350. This chimeric virus can be neutralized by 72A1 and infects rhesus macaques, providing a novel animal model for studying EBV gp350-specific reagents directly in vivo. Epithelial cell infection is mediated by binding of the EBV glycoprotein H and L complex to integrins. gH and gL are much more well conserved in rhLCV and a potent neutralizing mab, E1D1, that prevents epithelial cell infection cross-reacts with the integrin binding site on rhLCV gH/gL. These neutralizing mabs will be used to experimentally test the role of B and epithelial cell infection in acute and persistent infection after oral challenge withthe chimeric virus in rhesus macaques. An important strength of this proposal is its advancement of our basic understanding of EBV biology, as well as its immediate translational impact. Better understandings of the early events in EBV infection after oral transmission and experimental testing for the role of neutralizing antibodies targeting either B or epithelial cell infection proide a foundation for EBV vaccine development based on mechanism, rather than trial and error. In addition, these studies can provide important pre-clinical data to advance the use of neutralizing mabs as a therapeutic to prevent EBV-induced disease in immunosuppressed, EBV-naive hosts.